CN102112879B - STAT3 and TYK2 as drug targets for neurodegenerative diseases - Google Patents

Abstract

The invention provides Stat3 and Tyk2 targets that have importance for diagnosis of neurodegenerative diseases such as Alzheimer's disease. Stat3 and Tyk2 are also important as targets for drug development for neurodegenerative diseases.

Description

STAT3 and TYK2 is as the drug target of nerve degenerative diseases

Background technology

Nerve degenerative diseases is Alzheimer disease (AD) major effect the elderly such as.AD is a kind of PD, and it causes cognitive decline and dementia.Two pathologic marks of AD are cellular neurofibrillary entanglement (neuro fibrillary tangle, NFT) and extracellular senile plaque expellings (senile plaque, NP).The former accumulates in fast dead cell, and the latter sets up between neurocyte.Involve multiple process in nervus retrogression morbidity, comprise the generation of amyloid-beta, the hyperphosphorylation of tau, oxidative stress, inflammation, cynapse exhaustion and neuron and adjust and die.But the true inducement of AD is still waited to illustrate.A kind of hypothesis is thought, the neurone loss in AD is the accumulation due to toxic protein.Amyloid-beta is the cleaved products derived from amyloid precursor protein (APP), and it accumulates in AD brain as extracellular spot.Emerging research discloses: in specific cell, the imbalance of signal pathway causes the neuron of AD or A β-induction tune to be died.Such as, the activation of JNK with p38 relevant to mouse AD model people such as (, (2002) J Neurosci 22:3376-85) Savage is found.Also proved that glycogen synthase kinase (GSK)-3 approach participates in Neuronal cell death mechanism (people such as Koh, (2003) Brain Res Mol Brain Res 118:72-81 of oxidative stress induction; The people such as Takadera, (2004) EurJ Pharmacol.499:239-45).In addition, the suppression of GSK-3 reduces the neurotoxicity of A β-induction (people such as Koh, (2008) Brain Res 1188:254-62).Similarly, the intracellular signaling of both Wnt and Notch has certain effect (people such as De Strooper, (2001) J Cell Biol 152:F17-20 in AD morbidity; The people such as Anderton, (2000) Mol Med Today6:54-9).

At present, the clinical diagnosis of AD needs assessment medical history and health check-up, comprises neurology, Neuropsychology and psychiatric evaluation, and the test of various biology, radiology and electrophysiology.Although there is this succession of detection, definite diagnosis can only be reached by after death brain inspection.

Therefore, there are still unsatisfied needs, namely need a kind ofly to detect nerve degeneration in early days and monitor the simple biochemical test of relevant disease and disease progression.Aging populations, the such as U.S., Europe and Chinese aging populations seek the instrument of Diagnosis and Treat AD and other Neurodegenerative conditions improved just day by day.

The present invention relates to associating of another kind of signal pathway and AD illness, and relate to the qualification of two new target drones related in the molecule mechanism of AD, namely STAT3 albumen and TYK2, TYK2 are the upstream regulation agent of STAT3.STAT3 and TYK2 mainly involves in AD diagnosis and the exploitation for the medicine of this disease.Only show the process LAN of STAT3 and inflammation and related to cancer before.

STAT3 and upstream regulation agent TYK2 thereof all has Main Function in the neurotoxicity of A β-induction.In the cortex and hippocampus of AD brain, STAT3 strengthens in the phosphorylation of 705 tyrosine, and this can promote that the tune of cortical neuron is died, and TYK2 regulates phosphorylation and the activation of STAT3.One or two blocking-up in these targets can stop the cell death of A β-induction and can block progression of disease potentially.Therefore, the present invention exploitation treatment AD medicine and diagnosis this disease in significant.

Summary of the invention

In some embodiments, the invention provides the method and composition of the qualification regulation and control Neurodegenerative conditions such as compound of Alzheimer disease (AD).In some embodiments, said method comprising the steps of: compound contacts with STAT3 or TYK2 polypeptide by (i); (ii) measure the functional effect of this compound for STAT3 or TYK2, wherein said functional effect indicates this compound modulates Neurodegenerative conditions.In some embodiments, said method comprising the steps of: the polynucleotide of compound with coding STAT3 or TYK2 polypeptide contact by (i); (ii) measure the functional effect of this compound for STAT3 or TYK2, wherein said functional effect indicates this compound modulates Neurodegenerative conditions.Those skilled in the art will recognize that the method comprises contrast, such as, when there is not described compound, or when there is known activator compound, measuring expression or the activity of STAT3 and TYK2.

In some embodiments, the feature of Neurodegenerative conditions is the deposition of 4 amyloid in brain, such as Alzheimer disease (AD).In some embodiments, Neurodegenerative conditions is selected from AD, mild cognitive impairment, Parkinson's, repeats of dentatorubropallidolatrophy atrophy disease (DRPLA), transparent inclusion disease (NIHID) in neuronal kernel, dementia with Lewy body, Tang's grace syndrome, Hallervorden Spatz disease, prion disease, Argyrophilic grain dementia, cortical basal degeneration, dementia pugilistica, dispersivity neurofibrillary tangles, GSSShi disease (Gerstmann-Straussler-Scheinker disease), Hallervorden Spatz disease, Creutzfeldt-Jakob disease, Niemann-Pick disease 3 type, stein-leventhal syndrome, subacute sclerosing panencephalitis, spinocerebellar ataxia, Huntington disease, pik disease and repeats of dentatorubropallidolatrophy atrophy disease.

In some embodiments, polynucleotide or polypeptide are restructuring.In some embodiments, measurement function effect in vitro.In some embodiments, measurement function effect in eukaryotic such as neuronal cell.

In some embodiments, the expression by measuring TYK2 or STAT3 carrys out measurement function effect.In some embodiments, the determination method such as RT-PCR based on hybridization is used to measure expression.Or, use the determination method such as immunoassay based on albumen to measure expression.

In some embodiments, phosphorylation or kinase activity by measuring TYK2 carry out measurement function effect.In some embodiments, measurement function effect is carried out by the phosphorylation of phosphorylation such as 705 tyrosine measuring STAT3.In some embodiments, by measuring the activity of STAT3, such as Dimerized or downstream transcription activates measurement function effect.In some embodiments, to die measurement function effect by measuring the tune of cell.

In some embodiments, compound is selected from the specific antisense molecule of TYK2 or STAT3, RNAi molecule, antibody or antibody fragment, little peptide or organic molecule.

Present invention also offers the method and composition for diagnosis of neurodegenerative illness and disease.In some embodiments, said method comprising the steps of: (i) obtains biological sample from individuality; (ii) level of TYK2 polynucleotide or polypeptide in described sample is measured; (iii) level of TYK2 in the level of TYK2 in the sample of step (ii) and normal biological specimen is compared, thus diagnose the Neurodegenerative conditions in described individuality.

In some embodiments, determination step comprises measurement example based on hybridization as RT-PCR, or, based on the measurement example of albumen as immunoassays.In some embodiments, determination step comprises functional examination, such as kinase assays.In some embodiments, measure and compare the level of the Tyk2 of phosphorylation.

In some embodiments, said method comprising the steps of: (i) obtains biological sample from individuality; (ii) level of STAT3 polynucleotide or polypeptide in described sample is measured; (iii) level of STAT3 in the level of STAT3 in the sample of step (ii) and normal biological specimen is compared, thus diagnose the Neurodegenerative conditions in described individuality.

In some embodiments, determination step comprises measurement example based on hybridization as RT-PCR, or, based on the measurement example of albumen as immunoassays.In some embodiments, determination step comprises functional examination, such as, measures the dependent gene expression of STAT3.In some embodiments, the level of the STAT3 polypeptide of phosphorylation is measured, such as, at the STAT3 of 705 tyrosine phosphorylations.

In some embodiments, the feature of Neurodegenerative conditions is the deposition of 4 amyloid in brain, the brain of such as AD patient.In some embodiments, Neurodegenerative conditions is selected from Alzheimer disease (AD), mild cognitive impairment, Parkinson's, repeats of dentatorubropallidolatrophy atrophy disease (DRPLA), transparent inclusion disease (NIHID) in neuronal kernel, dementia with Lewy body, Tang's grace syndrome, Hallervorden Spatz disease, prion disease, Argyrophilic grain dementia, cortical basal degeneration, dementia pugilistica, dispersivity neurofibrillary tangles, GSSShi is sick, Hallervorden Spatz disease, Creutzfeldt-Jakob disease, Niemann-Pick disease 3 type, stein-leventhal syndrome, subacute sclerosing panencephalitis, spinocerebellar ataxia, Huntington disease, pik disease and repeats of dentatorubropallidolatrophy atrophy disease.

In some embodiments, relative to the Neurodegenerative conditions in the expression of TYK2 or STAT3 of normal specimens or activity level instruction individuality.In some embodiments, described method and other diagnostic method such as those conbined usage described herein.

Accompanying drawing explanation

In the cortex of Fig. 1: transgenosis AD mouse model and hippocampus, the tyrosine phosphorylation of STAT3 increases.A: cracking is from the cortex of the brain of 9 monthly age APP/PS1 (have Swedish suddenly change the APP695 of KM670/671NL and PS1 Δ E9) bi-transgenic mice, hippocampus and cerebellum and carry out Western blotting according to shown in figure.B: cracking from the brain of the APP/PS1 transgenic mice of different phase cortex and carry out Western blotting according to shown in figure.

The tyrosine phosphorylation of Fig. 2: in the cortical neuron of the cultivation stimulated with A β 25-35, STAT3 increases.Use A β 25-35 (40 μMs; H ₂o is used as solvent control) process the Primary cortical neurons of 7DIV, continue the time shown in figure.Collecting cell lysate also uses P-TyrSTAT3 and total STAT3 antibody to carry out Western blotting.

The neuroprotective unit that knocks out of Fig. 3: STAT3 dies from the tune of A β-induction.The cell death knocking out the PC12 cell decreasing A β 25-35 process induction of A:STAT3.Use STAT3siRNA transfection PC12 cell.The western blot analysis confirmation of STAT3 the knocking out of STAT3 in the STAT3siRNA transfection PC12 cell of 3 days, but use the PC12 cell mixing siRNA transfection then there is no knocking out (alpha-tubulin is used as loading control) of STAT3.With A β 25-35 by through the STAT3siRNA transfection PC12 cell process of 3 days 24 hours.By MTT determination method assessment cells viability, * represents p < 0.05.B: block the activation of STAT3 reduce A β 25-35 process after neuron in the increase of Caspase-3 of cutting.Before A β 25-35 process, STAT3 inhibitor peptide (200 μ g/ml) the pre-service cortical neuron of use cell permeability 30 minutes.Then use Caspase-3 antibody by immunoblotting assay cell lysate.The level of the Caspase-3 of cutting is carried out quantitatively.

Phosphorylation and the Neuronal cell death of Fig. 4: the STAT3 of beta amyloid inducing peptide need Tyk2.A: use various inhibitor by the Primary cortical neurons of pre-service shown in figure 2 hours, described inhibitor comprises the potent inhibitor (JAK inhibitor 1) of JAK, JAK2 inhibitor (AG490), Src tyrosine kinase inhibitor (PP2) and protein synthesis inhibitor (cycloheximide); Then A β 25-35 peptide process 4 hours or 24 hours are used.P-TyrSTAT3 and total STAT3 antibody on cell lysate is used to carry out Western blotting.B: use A β 25-35 process Primary cortical neurons.Use Tyk2 or JAK2 antibody on cell lysate to carry out immunoprecipitation, then carry out western blot analysis with phosphorylation-tyrosine (P-Tyr) antibody.C: use A β 25-35 process from Tyk2 ^-/-primary cortical neurons prepared by mouse.P-Tyr STAT3, total STAT3 and Caspase-3 antibody on cell lysate is used to carry out Western blotting.Quantitative test (plate below) is carried out to the multiple change of the STAT3 of tyrosine phosphorylation and the Caspase-3 of cutting.

Fig. 5: after using A β 25-35 process, the iNOS RNA in the transcriptional activity of the STAT3 in PC12 cell and the cortical neuron of cultivation raises.A. STAT1 (pGAS-Luc) or STAT3 (pSTAT3-Luc) reporter construct associating internal contrast plasmid (beta galactosidase-pCMV) transfection PC12 cell is used.Make Cell Differentiation 4 days by NGF, then use A β 25-35 or NGF (positive control) to process 1 day.Luciferase activity in working sample also corrects according to the activity of beta galactosidase.Result corrects according to the activity of beta galactosidase; Mean value ± S.E., n=3; *, p < 0.05.B. use the Primary cortical neurons 30 minutes of STAT3 inhibitor or JAK inhibitor I pre-service 7DIV, then use A β 25-35 (40 μMs) process 6 hours.Collect total serum IgE and carry out reverse transcription.Then iNOS primer pair cDNA is used to carry out real-time PCR analysis.

Fig. 6: in the hippocampus of patients with Alzheimer disease, STAT3 tyrosine phosphorylation increases.P-Tyr STAT3 antibody and neuronal specificity mark NeuN or glial specificity mark GFAP is used to be dyeed by the paraffin section of the brain of AD patient.The quantitative test that P-Tyr STAT3 dyes shows the common location of NeuN and GFAP.

Embodiment

The present invention establishes the STAT3 phosphorylation of increase and Neurodegenerative conditions associating such as between Alzheimer disease (AD).We find, the mark amyloid beta (A β) of AD induces the activation of STAT3, and the activation of this STAT3 be neuronal cell adjust die required.TYK2 is upstream regulation agent, the tyrosine phosphorylation of the STAT3 that A is beta induced in its mediation AD.

As in following examples prove, in the cortex showing the brain of the mouse model of AD illness multiple and hippocampus, the tyrosine phosphorylation of STAT3 unanimously raises.Also the STAT3 (that is, at the STAT3 of 705 tyrosine phosphorylations) of activation is detected in the brain region with senile plaque expelling (it is the deposition of A β).On the contrary, in the cortex of wild-type mice, then almost can't detect the tyrosine phosphorylation of STAT3, because herein illustrating the pTyr-STAT3 level of increase and associating between AD illness.

It is the association of STAT3 and A β at 705 tyrosine phosphorylations that another of STAT3 and AD contacts.Find phosphorylation and the activation of the beta induced STAT3 of A, it causes Neuronal cell death.In addition, when blocking the activation of STAT3, the beta induced cell death of A significantly reduces, and this illustrates that STAT3 activity has vital role in the Neuronal cell death that A is beta induced.

The present invention also comprises the effect of newfound TYK2 in AD illness, the tyrosine phosphorylation of the STAT3 that its mediation A is beta induced.In the cortical neuron being derived from TYK2 knock-out mice, the beta induced STAT3 of A reduces in the phosphorylation of 705 tyrosine.Therefore, our accumulation finds to identify STAT3 and TYK2 as the medicine developed for Neurodegenerative conditions and diagnostic core target.

A. define

" STAT3 " used herein refers to the member of intracellular signaling and transcription activator (STAT) protein family.STAT3 has two kinds of splice variants, called after STAT3 α (p92) and β (p83).An example of STAT3 polypeptide is the mankind STAT3 polypeptide described in Genbank registration number P40763.2, and its 705 amino acids is tyrosine.Exemplary STAT3 coded sequence is the human sequence described in Genbank registration number L29277.STAT3 also refer to have the species homologue of STAT3 signaling activity, splice variant, Polymorphic variant and STAT3 conservative property modify variant.

" TYK2 " used herein refers to the member of Janus kinases (JAK) family of tyrosine kinase.An example of TYK2 polypeptide is the human polypeptide sequence described in Genbank registration number P29597.3.Exemplary TYK2 coded sequence is the human sequence described in Genbank registration number X54637.TYK2 also comprise there is TYK2 tyrosine kinase activity species homologue, splice variant, Polymorphic variant and TYK2 conservative property modify variant.

Term " nerve degenerative diseases " and " Neurodegenerative conditions " alternatively use in this article mutually, refer to following illness, comprise: Alzheimer disease (AD), Parkinson's, repeats of dentatorubropallidolatrophy atrophy disease (DRPLA), transparent inclusion disease (NIHID) in neuronal kernel, dementia with Lewy body, Tang's grace syndrome, Hallervorden Spatz disease, prion disease, Argyrophilic grain dementia, cortical basal degeneration, dementia pugilistica, dispersivity neurofibrillary tangles, GSSShi is sick, Hallervorden Spatz disease, Creutzfeldt-Jakob disease, Niemann-Pick disease 3 type, stein-leventhal syndrome, subacute sclerosing panencephalitis, spinocerebellar ataxia, Huntington disease, pik disease and repeats of dentatorubropallidolatrophy atrophy disease.Usually the deposition being characterised in that neurofibrillary tangles in brain and/or 4 amyloid of Neurodegenerative conditions.

" diagnosis " of nerve degenerative diseases or illness refers to the symptom that prediction is relevant to these illnesss, and the A β of such as impaired cognitive function, accumulation deposits and Neuronal cell death.In some embodiments, cognitive function can be measured by conventional mini-mentalstate examination (Mini-Mental StateExam).In one embodiment, prognosis can be used to adjust treatment thus to provide better result.

" biological sample " comprises histotomy, such as biopsy samples (such as neuron biopsy) or the freezing microtome section for histology object.This type of sample also comprises blood and blood constitutent (such as serum, blood platelet, red blood cell etc.), celiolymph, cultured cells, the cell, ight soil, urine etc. of such as primary culture, explant and conversion.Usually biological sample is obtained from mammal such as primate or the mankind.

" normal biological specimen " used herein or " normal individual " represent baseline value or contrast." normally " representative does not suffer from the healthy individuals that Neurodegenerative conditions maybe can not suffer from Neurodegenerative conditions and (measures according to standard method, such as recognition tests or use biological sample, such as neuron biopsy or celiolymph) in polynucleotide or the level of nucleic acid.

" nucleic acid ", " nucleotide " or " polynucleotide " refer to deoxyribonucleotide or ribonucleotide, and their strand or the polymkeric substance of double chain form, and complement.This term comprises containing known nucleotide analog or the framework residue of modification or the nucleic acid of key, it is for synthesis, naturally occurring and non-natural existence, itself and reference nucleic acid have similar in conjunction with feature, and it is to be similar to the mode metabolism of reference nucleotide.The example of this kind of analog includes but not limited to, thiophosphate, phosphoramidate, methyl phosphonate, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide nucleic acid (PNA).

Unless otherwise, otherwise concrete nucleotide sequence also infers the variant (such as degenerate codon replacement) and complementary series that comprise the modification of its conservative property, and the sequence clearly indicated.Specifically, 3rd position of " wherein one or more are selected (or all) codons be replaced by the sequence of mixing base and/or deoxyinosine residue " degenerate codon can be realized replace (people such as Batzer, Nucleic Acid Res.19:5081 (1991) by producing; The people such as Ohtsuka, J.Biol.Chem.260:2605-2608 (1985); The people such as Rossolini, Mol.Cell.Probes8:91-98 (1994)).Term nucleic acid and gene, cDNA, mRNA, oligonucleotides and polynucleotide alternatively use mutually.

Term " polypeptide ", " peptide " and " albumen " alternatively use in this article mutually, and it refers to the polymkeric substance of amino acid residue.This term is applicable to such amino acid polymer: wherein one or more amino acid residues are corresponding naturally occurring amino acid whose manufactured chemical's analogies, is also applicable to the amino acid polymer of naturally occurring amino acid polymer and non-natural existence.

Term " amino acid " refers to amino acid that is naturally occurring and synthesis, and with the amino acid analogue being similar to naturally occurring amino acid whose mode effect and amino acid analog thing.Naturally occurring amino acid is the amino acid of being encoded by genetic code, and amino acid modified afterwards, such as hydroxyproline, Gla and O-phosphoserine.Amino acid analogue refers to the compound with naturally occurring amino acid with identical basic chemical structure, the carbon be namely combined with hydrogen, carboxyl, amino and R group, such as homoserine, nor-leucine, methionine sulfoxide, methionine methyl sulfonium.This kind of analog has the R group (such as nor-leucine) of modification or the peptide backbone of modification, but keeps the basic chemical structure identical with naturally occurring amino acid.But amino acid analog thing refers to that structure is different from amino acid whose general chemical constitution to be similar to the chemical compound of naturally occurring amino acid whose mode effect.

The monocase can recommended by its usually known three-character doctrine or IUPAC-IUB biochemical nomenclature commission herein censures amino acid.Similarly, nucleotide censured by the monocase code that usually can be accepted by it.

" variant that conservative property is modified " is applicable to both amino acid and nucleotide sequence.About concrete nucleotide sequence, the variant that conservative property is modified refers to the nucleic acid of the identical or substantially identical amino acid sequence of coding, or, when nucleic acid not encoding amino acid sequence time, this term refers to substantially the same sequence.Due to the degeneracy of genetic code, a large amount of functionally identical any given protein of nucleic acid coding.Such as, codon GCA, GCC, GCG and GCU all coded amino acid alanine.Therefore, in each position being appointed as alanine by codon, this codon can change over described any corresponding codon and not change coded polypeptide.This type of Nucleic acid variant is " silent variation ", and it is the one of conservative property modification variant.Each nucleotide sequence of coded polypeptide also describes the possible silent variation of often kind of this nucleic acid herein.Those skilled in the art will recognize that, and each codon in nucleic acid (except AUG, the unique codon of its normally methionine; And TGG, the unique codon of its normally tryptophane) can be modified to produce functionally identical molecule.Therefore, each silent variation hint of the nucleic acid of coded polypeptide is in each described sequence, and this is with regard to expression product, is not with regard to actual probes sequence.

About amino acid sequence, those skilled in the art will recognize that single replacement, deletion or the interpolation (it changes, increase or the single amino acids deleted in coded sequence or very little percentile amino acid) carried out for nucleic acid, peptide, polypeptide or protein sequence are " variants that conservative property is modified ", wherein said change causes amino acid to be replaced by chemically similar amino acid.It is well-known in the art for providing the conservative property of functionally Similar amino acids to replace form.The variant that this type of conservative property is modified is variant except Polymorphic variant of the present invention, interspecies homologs and allele, but the variant that conservative property is modified do not get rid of above-mentioned these.

Term " process LAN (overexpress) ", " process LAN (overexpression) " or " process LAN (overexpressed) " alternatively refer to mutually, for normal cell, with the albumen of detectable higher level transcription or translation or nucleic acid (RNA) in cell.This term comprises owing to transcribing, transcribing aft-loaded airfoil, translation, post translational processing, celluar localization (such as organelle, tenuigenin, nucleus, cell surface), and RNA and protein stability (for normal cell) and the process LAN caused.The routine techniques (i.e. RT-PCR, PCR, hybridization) detecting mRNA or the routine techniques (i.e. ELISA, immunohistochemistry technique) detecting protein can be used to be detected expression.Process LAN can be higher than normal cell by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher.In some instances, process LAN is high 1 times for normal cell, 2 times, 3 times, 4 times or higher transcribing or translation skill.

Term " low expression (underexpress) ", " low expression (underexpression) " or " low expression (underexpressed) " alternatively refer to mutually, for normal cell, with the albumen of detectable lower level transcription or translation or nucleic acid in cell.This term comprises owing to transcribing, transcribing aft-loaded airfoil, translation, post translational processing, celluar localization (such as organelle, tenuigenin, nucleus, cell surface), and RNA and protein stability (relative in contrast) and the low expression caused.The routine techniques (i.e. RT-PCR, PCR, hybridization) detecting mRNA or the routine techniques (i.e. ELISA, immunohistochemistry technique) detecting protein can be used to detect low expression.Low expression can be contrast low by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or lower.In some instances, low expression is relative to low 1 times, 2 times, 3 times, 4 times or lower transcribing or translation skill in contrast.

Term " differential expression " or " differential adjustment " generally refer to for other sample of at least one, and the albumen in a kind of sample or nucleic acid are process LAN (rise) or low expression (downward).For the purposes of the present invention, usually by come from suspect the sample of suffering from the individuality of Neurodegenerative conditions with come from known suffer from this illness (positive control) or known be that the sample of the individuality of feminine gender (negative control) compares to this illness.

" label " or " can detecting portion " be the composition that can be detected by spectrophotometric method, photochemical method, biochemical process, immuno-chemical method, chemical method or other physical method.Such as, useful label comprises 32P, fluorescent dye, electron-dense reagent, enzyme (in such as ELISA normally used enzyme), biotin, digoxin (digoxigenin) or haptens and can be prepared to detectable albumen (such as by mixing radioactive mark's thing in peptide) or the albumen for detecting the antibody reacted with peptide specific.Label can be bonded to targeted molecular, such as antibody or the nucleotide sequence for specific detection target compound.

When for censuring such as cell, nucleic acid, albumen or carrier, term " restructuring " representative is by importing heterologous nucleic acids or albumen or changing natural acid or albumen and cell, nucleic acid, albumen or carrier through modifying, or this is cell-derived from the cell through described modification.Therefore, such as, recombinant cell expresses non-existent gene in the cell of natural (non-recombinant) form, or express those unconventionality expressions, low expression or the natural gene of not expressing.

" antibody " refers to such polypeptide, and it comprises from specific recognition and the framework region of the immunoglobulin gene of conjugated antigen or its fragment.The immunoglobulin gene identified comprises κ, λ, α, γ, δ, ε and μ constant region gene, and numerous immune globulin variable region genes.Light chain is classified as κ or λ.Heavy chain is classified as γ, μ, α, δ or ε, and it in turn defines the classification of immunoglobulin (Ig) then, is IgG, IgM, IgA, IgD and IgE respectively.Usually, the antigen binding domain of antibody is most important for the specificity combined and compatibility.Antibody can be polyclonal or monoclonal, derived from serum, hybridoma or recombinant clone, can also be chimeric, clever long source or humanized.

Exemplary immunoglobulin (Ig) (antibody) structural unit comprises the tetramer.Each tetramer forms identical polypeptied chain by two, and often pair has one " light chain " (about 25kDa) and one " heavy chain " (about 50-70kDa).The N-end of every bar chain defines the amino acid whose variable region of about 100 to 110 or more, primary responsibility antigen recognizing.Term variable region of light chain (V _l) and variable region of heavy chain (V _h) refer to these light chains and heavy chain respectively.

Antibody exists with the form of such as intact immunoglobulins, or exists with the form of the fragment of the multiple clear sign produced by various peptidase digestion.Therefore, such as, pepsin digests antibody under the disulfide bond of hinge area, thus produces F (ab) ' ₂, it is the dimer of Fab, and Fab itself is by disulfide bond and V _h-C _h1 light chain connected.F (ab) ' ₂the disulfide bond destroyed in hinge area can be reduced in a mild condition, thus by F (ab) ' ₂dimer is converted into Fab ' monomer.Fab ' monomer be substantially there is part hinge district Fab (see Fundamental Immunology (Paul ed., 3d ed.1993).Although define Multiple Antibodies fragment according to the digestion of complete antibody, those skilled in the art will recognize that can by chemical mode or by using recombinant DNA method to synthesize this type of fragment from new (de novo).Therefore, term antibody used herein also comprises by carrying out whole antibody modifying the antibody fragment produced, or those antibody fragments using recombinant DNA method to use Phage displaying library to identify from the antibody fragment (such as scFv) of new synthesis or those (see, the people such as such as McCafferty, Nature 348:552-554 (1990)).

Term used herein " single-chain antibody " (scFv) refers to that in polypeptide connects, to comprise VH domain and VL domain (general such as, by introns peptide, [Gly-Gly-Gly-Gly-Ser] _xconnect) polypeptide, it can comprise the amino acid sequence outside two at amino and/or carboxyl terminal.Such as, single-chain antibody can comprise the tether section (tethersegment) for being connected to coded polynucleotide.As an example, scFv is single-chain antibody.The albumen that single-chain antibody is generally made up of one or more polypeptide section, described section be in fact by the gene code of immunoglobulin superfamily at least 10 continuous amino acids (such as, see TheImmunoglobulin Gene Superfamily, A.F.Williams and A.N.Barclay, in Immunoglobulin Genes, T.Honjo, F.W.Alt, and T.H.Rabbitts, eds., (1989) Academic Press:San Diego, Calif., pp.361-387, be incorporated to by reference herein), the most normally by rodent, non-human primates, bird, pig, ox, sheep, goat or human heavy chain or light chain gene sequential coding.Functional single chain antibodies comprises usually to be enough to keep the part to the immunoglobulin superfamily gene outcome of the binding characteristic of certain target molecules (normally acceptor or antigen (epi-position)).

" antibody " used herein can also refer to any functional VH and VL to (namely can specific binding epitope), it is connected to other polypeptide that can perform several functions separately with multiple configuration, such as the stabilizing agent of acceptor, acceptor inhibitor or VH-VL compound.

" immune globulin variable region domain " used herein refers to any VH or the VL domain as bound fraction (without VH or VL domain).With regard to antibody, this type of domain can be connected to other polypeptide that can perform several functions with multiple configuration, such as acceptor, acceptor inhibitor or reporter molecule activator.

When censuring albumen, nucleic acid, antibody or micromolecular compound, word " specificity (or selectivity) combination " refers to association reaction, and whether it is determined in the heterogeneous population of albumen or nucleic acid and other biopreparate usually exists albumen or nucleic acid (such as STAT3, TYK2 or its modified forms).When antibody, under the immunoassay conditions of specifying, specify the combination of antibody and specific protein can be at least 2 times of background, more than 10 times to 100 times of more generally background.Need with the specific binding of antibody in such a situa-tion those with regard to its for the selectivity of specific protein by the antibody selected.Such as, polyclonal antibody can be screened only to obtain those and selected antigen has specific immune response and do not have the polyclonal antibody of specific immune response with other albumen.This screening can have the antibody of cross reaction to realize with other molecule by cutting.Panimmunity mensuration form can be used to select to have with specific protein the antibody of specific immune response.Such as, solid phase ELISA immunoassays are conventionally used in selects to have the antibody of specific immune response (see such as Harlow & Lane with albumen, Antibodies, ALaboratory Manual (1988), which depict the form and condition that can be used for the immunoassays measuring specific immune response).

In the mensuration regulation and control compound of AD or the mensuration of nerve degeneration biomarker, word " functional effect " comprises the parameter measuring indirectly or directly affect by biomarker of the present invention, such as chemistry or phenotypic effect.Therefore, functional effect comprises intracellular signaling event at once, such as transcriptional activation or phosphorylation, or more indirectly effect, such as gene expression or Apoptosis." functional effect " comprises external, in body and in vitro activity.

" measurement function effect " is meant to measure the compound raising or reduce the parameter that those indirectly or directly affect by biomarker of the present invention, such as mensuration physics and chemistry or phenotypic effect.This type of functional effect can be measured by any method known to those skilled in the art, such as, by the change (such as, fluorescence, absorption, refractive index) of spectroscopic properties; Fluid mechanics (such as shape), chromatography; Or the solubility characteristics of albumen; Measure the transcriptional activation of derivable mark or mark; Measure the change that enzyme (such as kinases) is active; Increase or reduce the ability of cell proliferation, apoptosis, cell-cycle arrest; Measure the change of cell surface marker.Evaluation function effect can be carried out by a lot of method well known by persons skilled in the art, such as microscopic method that the is quantitative or change of qualitative determination morphological feature, measure the RNA of other gene or the change of protein level of cells, measure rna stability, qualification downstream or reporter molecule gene expression (CAT, luciferase, β-gal, GFP etc.), such as, combined by chemiluminescence, fluorescence, chrominance response, antibody, can induction sign thing etc.

" activity " of nerve degeneration biomarker polypeptide refers to the structure of the polypeptide in its n cell or tissue, adjustment or biochemical function.The activity of nerve degeneration biomarker comprises both direct activity and indirect activity.Exemplary direct activity is catalytic activity, i.e. the kinase activity of TYK2.The direct activity of the STAT3 of phosphorylation comprises Dimerized and transcriptional activation.Exemplary indirect activity is the character mutation of cell or tissue or response, such as apoptosis that produce for the direct activity of polypeptide.Catalytic activity can be measured by the amount such as measuring the substrate be phosphorylated.Methods known in the art can be used to assess other activity, such as apoptosis.

" inhibitor ", " activator " and " correctives " of mark is used in reference to the activation of the in vitro and in vivo determination method qualification using nerve degeneration biomarker (such as the STAT3 of STAT3, TYK2, phosphorylation, the TYK2 of phosphorylation), suppression or Molecular regulator." inhibitor " be such as combine, part blocks or block activity, reduction, prevention, delay activation, deactivation completely, go quick or lower the activity of nerve degeneration biomarker or the compound of expression." activator " is the compound of the activity increasing, open, activate, promote, strengthen activation, sensitization, excitement or raise nerve degeneration biomarker, such as activator.Inhibitor, activator or correctives also comprise the nerve degeneration biomarker through genetic modification, such as there is the form of the activity of change, and naturally occurring and that synthesize part, antagonist, activator, antibody, peptide, cyclic peptide, nucleic acid, antisense molecule, ribozyme, RNAi and siRNA molecule, organic molecule etc.This type of determination method of inhibitor and activator comprises, and such as in vitro, expresses nerve degeneration biomarker in cell or cell extract, uses the correctives compound of supposition, then measures functional effect for activity, as mentioned above.

By through potential activator, inhibitor or correctives process the sample comprising nerve degeneration biomarker (such as the STAT3 of TYK2, STAT3, phosphorylation, the TYK2 of phosphorylation) or measure and compare with the control sample not containing inhibitor, activator or correctives the degree changed with detection of active.Control sample (undressed or through known correctives process) is assigned to the relative protein activity value of 100%.When the activity value relative to contrast is about 80%, preferably 50%, more preferably during 25-0%, reach the suppression for nerve degeneration biomarker.When the activity value relative to contrast (without activator process) is 110%, more preferably 150%, more preferably 200-500% (namely high 2 to 5 times relative to contrast), more preferably during high 1000-3000%, reaches the activation for nerve degeneration biomarker.

Term " test compounds ", " test agent ", " correctives " or equivalent terms are in this article for describing any molecule that it directly or indirectly regulates the ability of nerve degeneration biomarker to be measured, it no matter is naturally occurring or synthesis, such as protein, (such as length is about 5 to about 25 amino acid to oligopeptides, preferably, length is about 10 to 20, or 12 to 18 amino acid, preferably, length is 12, 15 or 18 amino acid), organic molecule, polysaccharide, peptide, cyclic peptide, fat, fatty acid, siRNA, polynucleotide, oligonucleotides etc.Test compounds can be the form in the library of test compounds, such as, provide abundant multifarious combinatorial libraries or random library.Test compounds is optionally connected to fusion partner, such as target compound, redemption compound (rescue compound), Dimerized compound, stabilization compound, addressable compound (addressable compound) and other funtion part.Easily, be tested and appraised the test compounds (being called " lead compound ") with some characteristic wanted or activity (such as inhibit activities), the variant producing described lead compound, the characteristic evaluating these variant compound and activity and produce the new chemical entities with useful quality.Usually, apply high flux screening (HTS) and carry out this alanysis.

Term " organic molecule " and " Small molecular " refer to organic molecule that is naturally occurring or synthesis, it has more than about 50 dalton and molecular weight below about 2500 dalton, preferably approximately below 2000 dalton, preferably approximately 100 to about 1000 dalton, and more preferably about 200 to about 500 dalton.

B.STAT3 and TYK2

STAT3 belongs to intracellular signaling and transcription activator (STAT) protein family.The same with other member of this family, it is activated by phosphorylation by the kinases of receptors bind.Once be phosphorylated, namely stat protein forms homodimer or heterodimer, and described dimer is indexed into nucleus, and they play a role as transcription activator there.Except tyrosine phosphorylation that is Dimerized and that be indexed into needed for nucleus, stat protein also needs serine phosphorylation to carry out best transcriptional activation people such as (, (1995) Science 267:1990-94) Zhang.

In response to the stimulation of growth factor (such as epidermal growth factor, CSF-1 and PDGF) and multiple interferon and cell factor (such as IFN α, IL-6, LIF), STAT3 is activated (Akira (2000) Oncogene 19:2607-11).Being activated by the STAT3 of the cell factor of IL-6 family causes several functions to respond.After stimulation, cell factor is combined to cause Janus kinases (JAK) to the phosphorylation of STAT3 with gp130 and other associated receptor.Phosphorylation occurs in the amino acid whose single tyrosine site of 705 in transactivation domain, and this causes Dimerized, the transposition of STAT3 and DNA to combine (Levy and Lee (2002) J ClinInvest 109:1143-48).

To be activated in response to a large amount of signal transduction molecules due to it and to mediate multiple different signal pathway, so the function of STAT3 is ubiquity (namely its effect can cause propagation, promotes survival or cause apoptosis).Based on the report much delivered, its function comprises: acute stage in the inducing hepatocyte cancer cell response (people such as Alonzi, (2001) Mol Cell Biol21:1621-32), stimulate B lymphocyte proliferation, activated mononuclear Cell Differentiation and the retarded growth (people such as Heinrich, (1998) Biochem J 334:297-314), maintain the versatility (people such as Raz of embryonic stem cell, (1999) Proc Natl Acad Sci USA 96:2846-51), and the effect (people such as Moh in liver regeneration, (2007) Lab Invest 87:1018-28).Also find that the transcriptional activity of STAT3 is for neuronal cell survival significant (Battle andFrank (2002) Curr Mol Med 2:381-92).

Tyk2 belongs to the JAK family of intracellular protein-tyrosine kinase (PTK), and it comprises JAK1, JAK2 and JAK3.These PTK have the kinase domain of two arranged in series: carboxyl terminal, functional tyrosine kinase (TK) domain and contiguous kinases sample (KL) domain.JAK is played a significant role in intracellular signaling by various kinds of cell acceptor, as at first by its supplement for interferon (IFN) without the genetic defect of the mutational cell line of response ability prove.Found afterwards, induced the tyrosine phosphorylation of one or more JAK family member with the ligand binding of nearly all known cytokine receptor.These PTK make the sub-molecular phosphorus acidifying of downstream effect, and are vital for correct ligand-receptor intracellular signaling.

C. general recombination method

In order to the gene of expression cloning, the such as cDNA of coding STAT3 or TYK2, coded sequence subclone is entered expression vector by people usually, described expression vector comprise instruct transcribe promoter, transcribe/translation termination and for the ribosome bind site of translation initiation.Suitable promoters is well known in the art, sees the description of the people such as people and Ausubel (seeing above) such as such as Sambrook.Bacterial expression system for expressing CMR1 albumen can obtain in such as Escherichia coli, Bacillus (Bacillus sp.) and the Salmonella (Salmonella) (people such as Palva, Gene 22:229-235 (1983); The people such as Mosbach, Nature302:543-545 (1983)).Kit for this type of expression system can obtain from commercial channel.Eukaryotic expression system for mammalian cell, yeast and insect cell is well known in the art, also can obtain from commercial channel.Retroviral expression system also can be used in the present invention.

Specific application is depended in the selection being used to guide the promoter that heterologous nucleic acids is expressed.Preferably, the distance of promoter distance heterologous transcription starting point approximates greatly the distance of its distance transcripting start point in its natural surroundings.But, it is well known in the art that some changes of this distance can be accepted and do not lose promoter function.

Except promoter, expression vector comprises transcriptional units or expression cassette usually, and it is included in other elements all needed for nucleic acid of expressing coding STAT3 or TYK2 in host cell.Therefore, typical expression cassette comprises the promoter that is operably connected with coded sequence and signal, ribosome bind site and translation termination needed for the effective poly-adenosine of transcript.The other element of box can comprise enhancer, if use genomic DNA as structural gene, can also comprise the introne with functional splice donor and acceptor site.

Except promoter sequence, expression cassette also should comprise the transcription termination region being positioned at structural gene downstream, to provide effective termination.Terminator can obtain from the homologous genes obtaining promoter sequence, or can obtain from different genes.

Not crucial especially for hereditary information being passed to the particular expression carrier of cell.Any conventional carrier for expressing in eucaryon or prokaryotic can be used.The bacterial expression vector of standard comprises plasmid, such as, based on plasmid, pSKF, pET23D of pBR322, and amalgamation and expression system, such as MBP, GST and LacZ.Can also add epitope tag to provide conventional separation method in recombinant protein, described label is such as c-myc.Sequence label can be comprised to carry out nucleic acid redemption at expression cassette.Can comprise mark in the carrier, such as fluorescin, green or red fluorescent protein, β-gal, CAT etc., they are as the mark of carrier transduction.

The expression vector comprised from the controlling element of eukaryotic viral is generally used for carrier for expression of eukaryon, such as SV40 carrier, papilloma viral vector, retroviral vector and the carrier derived from Epstein-Barr virus.Other exemplary eukaryotic vector comprises pMSG, pAV009/A ⁺, pMTO10/A ⁺, pMAMneo-5, baculoviral pDSVE, and any other allows the carrier of expressing protein under the guidance of following promoter: CMV promoter, SV40 early promoter, SV40 late promoter, metallothionein promoter, MuMTV promoter, rous sarcoma virus promoter, polyhedrin promoter or other demonstrate for the effective promoter of the expression in eukaryotic.

The protein expression that inducible promoter regulates and controls to carry out from eukaryotic vector can also be used.Use inducible promoters, by introducing in promoter in response to the element of reagent (such as tetracycline or moulting hormone), make expression be associated with the concentration of these derivants.Generally speaking, only when there is derivant, obtain high-caliber expression from inducible promoters; Basal level expression is extremely low.

Standard transfection methods is used to produce bacterium, mammal, yeast or insect cell line, they express selected by albumen, then by use albumen described in standard technique purifying (see, the people such as such as Colley, J.Biol.Chem.264:17619-17622 (1989); Guide toProtein Purification, in Methods in Enzymology, vol.182 (Deutscher compiles, 1990)).According to standard technique carry out eucaryon and prokaryotic conversion (see, such as Morrison, J.Bact.132:349-351 (1977); Clark-Curtiss & Curtiss, Methods in Enzymology 101:347-362 (people such as Wu compiles, 1983).

Any program for exogenous nucleotide sequence being imported host cell known can be used.These comprise use calcium phosphate transfection, polybrene (polybrene), protoplast fusion, electroporation, biological bullet (biolistics), liposome, microinjection, plasma carrier, viral vectors and any genomic DNA, cDNA, the DNA of synthesis or the method (see people such as such as Sambrook, seeing above) of other exogenous genetic material importing host cell for cloning that other is known.Concrete genetic engineering program used only needs can successfully by least one channel genes host cell.

After expression vector transfered cell, be conducive to the cell cultivating transfection under the condition expressing institute's sortilin (such as TYK2 or STAT3).

D. diagnostic method

The invention provides prediction, diagnosis of neurodegenerative illness such as AD method or provide the method for prognosis for it, described method is by detecting the expression of STAT3 and TYK2 or activation.Prediction and prognosis comprise mensuration from the level of nerve degeneration biomarker expression or activation in the biological sample of individuality, then by this level with contrast baseline or scope compares.Typically, baseline value representative does not suffer from the polynucleotide of healthy individuals or the level of nucleic acid that maybe can not suffer from Neurodegenerative conditions (measuring by using biological sample such as neuronal cell biopsy or humoral sample such as celiolymph).The expression of nerve degeneration biomarker or the change of activation level can indicate this patient to have the risk of neurodegenerative increase.These class methods can with other diagnostic method conbined usage known in the art.

1. based on the mensuration of nucleotide

In some embodiments, the RNA of biological sample is used to detect the expression of STAT3 or TYK2 by real-time or quantitative PCR.RNA can be extracted by any method known to those skilled in the art, such as, use Trizol and RNeasy.PCR in real time can be carried out by any method known to those skilled in the art, such as, use the Taqman PCR in real time of AppliedBiosystem determination method.Calculate gene expression relative to the normal lung RNA collected, and this expression is corrected for housekeeping gene.Suitable Oligonucleolide primers is selected by those skilled in the art.

In one embodiment, nucleic acid binding molecule such as probe, oligonucleotides, oligonucleotide arrays and primer is used to detect RNA biomarker, to detect the differential rna expression in Patient Sample A.In one embodiment, RT-PCR is used according to standard method known in the art.In another embodiment, quantitative PCR assay can be used (such as can to obtain from such as Applied Biosystems determination method) detect nucleic acid and variant thereof.In other embodiments, nucleic acid microarray can be used to detect nucleic acid.Routine techniques such as northern can be used to analyze the analysis realizing nucleic acid, or any other is also included within scope of the present invention based on the method (such as slot blot hybridization) with nucleic acid array hybridizing (described nucleic acid array complementation is in a part for mark coded sequence).The reagent that can be combined with the biological nucleic acid mark selected according to method known to those skilled in the art preparation, or buy from commercial channel.

Applicable pcr amplification technology is described in the people such as people and Innis (seeing above) such as such as Ausubel.General nucleic acid hybridization is described in Anderson, " Nucleic AcidHybridization, " BIOS Scientific Publishers, 1999.Amplification or the hybridization of multiple nucleotide sequence (such as genomic DNA, mRNA or cDNA) can also be carried out from the mRNA be arranged in microarray or cDNA sequence.The generality of microarray method describes sees Hardiman, " Microarrays Methods and Applications:Nuts & Bolts, " DNA Press, 2003; With people such as Baldi, " DNA Microarrays and GeneExpression:From Experiments to Data Analysis and Modeling, " Cambridge University Press, 2002.

2. based on mensuration and the immunoassays of albumen

In another embodiment, any means in panimmunity determination method well known by persons skilled in the art can be used, in mensuration, use antibody reagent to detect expression and/or the activity level of nerve degeneration biomarker of the present invention in biological sample.The generality of immunoassay and program describes sees Price and Newman, " Principles and Practice ofImmunoassay, " 2nd Edition, Grove ' s Dictionaries, 1997; And Gosling, " Immunoassays:A Practical Approach, " Oxford University Press, 2000.Panimmunity determination techniques can be used, comprise competitiveness and non-competitive immunoassay.See, the people such as such as Self, Curr.Opin.Biotechnol., 7:60-65 (1996).The technology that term " immunoassays " is contained includes but not limited to, enzyme immunoassay (EIA) (EIA), such as enzyme amplification immunoassay (EMIT), enzyme linked immunosorbent assay (ELISA) (ELISA), IgM antibody catch ELISA (MAC ELISA) and microparticle enzyme immunoassay (EIA) (MEIA); Capillary Electrophoresis immunoassays (CEIA); Radiommunoassay (RIA); Immune radiating measures (IRMA); Fluorescence polarization immunoassays (FPIA); With chemical luminescent detecting (CL).If necessary, this type of immunoassay can carry out robotization.Immunoassays can also with the fluorescence conbined usage of induced with laser.See, the people such as such as Schmalzing, Electrophoresis, 18:2184-93 (1997); Bao, J.Chromatogr.B.Biomed.Sci., 699:463-80 (1997).Liposome immunization measures, and such as streamer penetrates liposome immunization mensuration and Liposome Immunosensor is also suitable for the present invention.See, the people such as such as Rongen, J.Immunol.Methods, 204:105-133 (1997).In addition, turbidimetry for Determination is also suitable in method of the present invention, and wherein, the formation of protein/antibody complex causes the light scattering of increase, and it is converted into the function of peak speed signal as marker concentration.Turbidimetry for Determination by commercial channel from Beckman Coulter (Brea, CA; Kit#449430) obtain, and Behring Nephelometer Analyzer people such as (, J.Clin.Chem.Clin.Biochem., 27:261-276 (1989)) Fink can be used to carry out.

Can in determination method described herein, use detectable part directly or indirectly to detect.Can use multiple can detecting portion, the selection of label is depended on required sensitivity, is specified (disposal provision) with easy degree, durability requirements and the available instrument of antibody conjugate and processing.Suitable can detecting portion include but not limited to, radioactive nucleotides, fluorescent dye (such as fluorescein, fluorescein isothiocynate (FITC), OregonGreen ^tM, rhodamine, Texas red, tetramethyl rhodamine isothiocyanate (TRITC), Cy3, Cy5 etc.), fluorescent marker (such as, green fluorescent protein (GFP), phycoerythrin etc.), the proteinase activated automatic quench fluorescence compound of being correlated with by tumour, enzyme (such as, luciferase, horseradish peroxidase, alkaline phosphatase etc.), nano particle, biotin, digoxin, metal etc.

The chemiluminescence assay of the chemiluminescence antibody of nucleic acid specificity is used to be suitable for carrying out sensitivity, the non-radioactive detection of protein level.With fluorophore mark antibody be also applicable.The example of fluorophore includes but not limited to DAPI, fluorescein, Hoechst 33258, R-phycocyanin, B-phycoerythrin, R-PE, rhodamine, Texas red and Liz amine (lissamine).Indirectly label comprises various enzyme well known in the art, such as horseradish peroxidase (HRP), alkaline phosphatase (AP), beta galactosidase, urase etc.Horseradish peroxidase detection system can use together with such as chromophoric substrate tetramethyl benzidine (TMB), and it produces the soluble product that can detect at 450nm when there is hydrogen peroxide.Alkaline phosphatase detection system can use together with such as chromophoric substrate para-nitro-pheneye phosphate, the soluble product that its generation easily can detect at 450nm.Similarly, beta galactosidase detection system can use together with chromophoric substrate ortho-nitrophenyl-β-D-galactopyranoside (ONPG), the soluble product that its generation can detect at 410nm.Urase detection system can with substrate such as urea-bromcresol purple (Sigma Immunochemicals; St.Louis, MO) use together.

Signal from direct or indirect label can be analyzed like this, such as: use spectrophotometer detection from the color of luminous substrate; Use radiation counter to detect radiation, such as, use gamma counter to detect ¹²⁵i; Or use photofluorometer to detect fluorescence when there is the light of certain wavelength.For the detection of enzyme len antibody, spectrophotometer such as EMAXMicroplate Reader (Molecular Devices can be used; Menlo Park, CA) carry out quantitative test according to the instructions of manufacturer.If necessary, mensuration of the present invention can robotization or by robot manipulation, and can detect the signal from multiple sample simultaneously.

Antibody can be fixed on many kinds of solids carrier, the surface (such as microtiter well) of such as magnetic or chromatography matrix particle, assay plate, multi-disc solid base material or film (such as plastics, nylon, paper) etc.Detector bar (assay strip) can be prepared by antibody or multiple antibody being coated on the array on solid carrier.Then this bar submergence also can be processed rapidly to produce measurable signal by washing and detecting step in the test sample, such as coloured speckle.

Useful physical form comprises, and has the surface of multiple discontinuous, addressable position, to detect multiple different mark.This type of form comprises microarray and some capillary equipment.See, the people such as such as Ng, J.Cell Mol.Med., 6:329-340 (2002); U.S. Patent number 6,019,944.In these embodiments, each noncontinuous surface position can comprise antibody to fix one or more marks, to detect in each position.Or surface can comprise one or more discontinuous particle (such as microparticle or nano particle), they are fixed on the discontinuous position on surface, and wherein said microparticle comprises antibody to fix one or more marks to detect.

Can be analyzed by multiple physical form.Such as, microtiter plate or automation equipment can be used for the process that promotes to test sample in a large number.Or, simple sample form can be developed to promote to carry out in real time diagnosing or prognosis.

Or antibody of the present invention or nucleic acid probe can be applicable in the section of the patient biopsy samples be fixed on microslide.Any one in multiple optics known in the art or fluorescence microscopy can be used to show antibody staining or the in situ hybridization pattern of generation.

In another form, various mark of the present invention additionally provides the reagent for in-vivo imaging, the image of such as labelled reagent, and it detects the nucleic acid of biomarker of the present invention or the albumen of coding.

Non-invasive medical imaging technique, such as PET (positron emission tomography) (PET) or single photon emission computerized tomography,SPECT (SPECT) imaging are useful especially for detection cerebral disease.The chemical functional of PET and SPECT imaging display official and tissue, other imaging technique, such as X-ray, CT and MRI then display structure.The use of PET and SPECT imaging strengthens just day by day in the qualitative effect with monitoring in the progress of cerebral disease such as AD.In some cases, PET or SPECT imaging is used to allow to detect Neurodegenerative conditions before the several years appears in symptom.

This strategy is for developing the compound of the in-vivo imaging of the amyloid beta deposition be suitable in mankind's brain.When testing in the patient suffering from AD, brain is for being limited for the monoclonal antibody of A β and the picked-up of fragments of peptides.Up to now, the Small molecular method for amyloid imaging is the most successful, and such as Nordberg A is shown in its description, Lancet Neurol., 3 (9): 519-27 (2004); Kung MP et al, Brain Res., 1025 (1-2): 98-105 (2004); The people such as Herholz K, Mol Imaging Biol., 6 (4): 239-69 (2004); NeuropsycholRev., the people such as Zakzanis KK, 13:1-18 (2003); Herholz K, Ann Nucl Med., 17:79-89 (2003).

Diagnostic compounds disclosed herein or its fragment can be used in the environment of PET and SPECT imaging applications.In order to PET or SPECT application, after modifying with suitable spike residue, diagnostic compounds (such as antibody fragment, itself and nerve degeneration biomarker protein interact) can be used to show the level of these biomarkers in individuality.This type of detection is the indication of the position of the characteristic neurological toxicity amyloid beta deposition of AD brain.

3. functional examination

The expression or the activity that detect nerve degeneration biomarker can also be measured by using function.In view of the known activity (such as tyrosine kinase) of TYK2 and the known activity (Dimerized, transcriptional activation) of STAT3 and disclosed herein those are active, this type of mensuration is apparent for those skilled in the art.Be determined in lower part, there is more detailed discussion for what detect the activity of nerve degeneration biomarker.

4. other diagnostic method

Method for diagnosis of neurodegenerative illness be known in the art (see, such as Diagnostic and Statistical Manual of Mental Disorders, FourthEdition (DSM-IV-TR), American Psychiatric Assoc.2000).Generally speaking, doctor or neurologist will consider multiple factor when diagnosing particular individual or patient.Such as, family history is often the indication of AD and other Neurodegenerative conditions risk.Doctor will carry out test chemical to check normal blood counting, thyroid function, liver function, glucose level.A part for the normally this test of analysis of spinal fluid.

Doctor can also use Neuropsychology test come assess memory, deal with problems, notice, vision coordination and abstract thinking.These comprise space exercise and simple calculating.Mini-mentalstate examination is also general.

Cat scan and MRI also can be used for getting rid of tumour, and can provide the clue about brain degradation regions.

E.STAT3 and TYK2 correctives compound

The reagent that correctives as nerve degeneration biomarker carries out testing can be any little chemical compound, or biological entities, such as protein, sugar, nucleic acid or fat.Usually, test compounds will be little chemical molecular, peptide, oligonucleotides or antibody fragment.

1. antibody and antibody fragment

In some embodiments, antibody or its epitope binding fragments can be used for suppressing STAT3 and TYK2 active.Known in the art have several to the antibody of these protein-specifics, and can obtain from commercial channel (see, such as Invitrogen products catalogue, can obtain from website invitrogen.com; And the products catalogue of BD Biosciences, can obtain from website bdbiosciences.com).

Or, standard technique can be used to produce TYK2 or STAT3 antibody (such as, see, Kohler & Milstein, Nature 256:495-497 (1975) from new (de novo); The people such as Kozbor, Immunology Today 4:72 (1983); The people such as Cole, pp.77-96inMonoclonal Antibodies and Cancer Therapy (1985)).Technology (United States Patent (USP) 4,946,778) for the production of single-chain antibody is applicable to the antibody produced for polypeptide of the present invention.In addition, transgenic mice or other biosome such as other mammal, can be used for expressing humanized antibody.Or, phage display technology can be used for identifying the antibody of antigen selected by specific binding and different poly-Fab fragment (see, the people such as such as McCafferty, Nature348:552-554 (1990); The people such as Marks, Biotechnology 10:779-783 (1992)).

Generally speaking, antibody inhibition comprises the specific antibody fragment kept for antigen or epi-position (such as the STAT3 of TYK2, STAT3, phosphorylation, the TYK2 of phosphorylation).The Functional domains of the usual targeting antigen of antibody inhibition, such as, the kinase domain of TYK2 or the Dimerized domain of STAT3.

In some embodiments, antibody inhibition comprises single-chain antibody, such as scFV.In some embodiments, antibody inhibition comprises Fab region.In some embodiments, antibody inhibition is humanized.

2. Nucleic acid inhibitors

The reagent (such as antisense molecule, ribozyme, DNA enzymatic, little inhibitory RNA etc.) that correctives also comprises the level being designed to the mRNA reducing encoding nerve degeneration biomarker polypeptide or the reagent (such as translate retarding agent, such as, be complementary to the antisense molecule of other sequence on translation starting point or mRNA molecule) reduced from the translation skill of mRNA.In some embodiments, known method is used to identify the nucleotide sequence suppressing nerve degeneration biomarker.This type of inhibitor can include but not limited to, siRNA oligonucleotides and antisense oligonucleotides.Inhibitor siRNA or antisense oligonucleotides can be used for target TYK2 and STAT3 specifically and express.

In some embodiments, RNA interference is used to produce little double-stranded RNA, little RNA interfering (siRNA) or short hairpin RNA (shRNA) inhibitor are to affect the expression of TYK2 and STAT3, and this is generally by cutting and destroy its cognate rna to carry out.The double-stranded RNA of little RNA interfering (siRNA or shRNA, the two alternatively uses mutually) normally 19-22nt.SiRNA can be obtained by chemosynthesis or by the RNAi technology based on DNA vector.Use the siRNA technology based on DNA vector, little DNA intron (about 70bp, the short hairpin RNA of its coding target target gene) clone is entered commercially available carrier.The carrier comprising intron transfection can enter cell, and expresses described short hairpin RNA.Hairpin RNA is processed into rapidly the double-stranded RNA (siRNA) of 19-22nt by cell mechanism.SiRNA is generally inserted in suitable RNAi carrier, because the siRNA of synthesis preparation tends to have lower stability and so ineffective in transfection.

Can use such as below with reference to the method described in document and algorithm to prepare the people such as siRNA:Wang, (2004) A Web-based Design Center for Vector-basedsiRNA and siRNA cassette Bioinformatics. (In press); The people such as Khvorova, (2003) Cell.115 (2): 209-16; The people such as Harborth, (2003) Antisense NucleicAcid Drug Dev.13 (2): 83-105; The people such as Reynolds, (2004) Nat Biotechnol.22:326-30; And the people such as Ui-Tei, (2004) Nucleic Acids Res.32:936-48.Be online instrument for building other instrument of siRNA sequence, such as siRNA TargetFinder and Construct Builder, can obtain from GenScript; Oligo Design andAnalysis Tools, can obtain from Integrated DNA Technologies; Or siDESIGN ^tMcenter, can obtain from Dharmacon, Inc..Suggestion uses ORF (opening code-reading frame) as target selection area, and 50-100nt place, preferred initiation codon downstream, builds siRNA.

3. peptide and micromolecular inhibitor

The reagent carrying out testing as the correctives of polypeptide of the present invention or polynucleotide can be any little chemical compound or biological entities, such as albumen, sugar, nucleic acid or fat.Test compounds generally includes little chemical molecular and peptide.Inhibitor peptides can be designed as native ligand or the binding partners of the molecule of simulation institute target.In the context of the present invention, the inhibitor peptides of exemplary STAT3 can design from the Dimerized companion of such as STAT3.The inhibitor peptides of exemplary TYK2 can design from the natural substrate of such as TYK2.

Substantially any chemical compound can be used as the potential correctives in mensuration of the present invention, although the most often use the compound of water soluble or organic (especially based on DMSO's) solution.Measure and be designed to carry out determination step by robotization and originate as mensuration provides compound to screen large chemistry library easily from any, usually (such as, carrying out with the form of microtitration on microtiter plate in automatic assay) is run abreast.To recognize, there is the supplier of a lot of chemical compound, comprise Sigma (St.Louis, MO), Aldrich (St.Louis, MO), Sigma-Aldrich (St.Louis, MO), FlukaChemika-Biochemica Analytika (Buchs, Switzerland) etc.

In one embodiment, high-throughput screening method comprises provides combinatorial chemistry or peptide library, and it comprises a large amount of potential therapeutic compound (potential correctives compound).Then according to description herein, in one or more mensuration, this type of " combinatorial chemistry library " or " peptide library " is screened, to identify those library constructs demonstrating the characteristic activities wanted (especially chemical species or subclass).The compound identified thus self can be used as therapeutic agent that is potential or reality as " lead compound " of routine or they.

Combinatorial chemistry library is the set of the number of chemical compound generally produced by chemosynthesis or biosynthesizing, realizes by combining multiple chemistry " structure module ".Such as, such as, by combining one group of chemical building blocks (amino acid) in often kind of possible mode for given compound length (amino acid number namely in polypeptide compound) and form linear combinatorial chemical library, polypeptide libraries.Millions of chemical compound can be synthesized by this type of combined hybrid of chemical building blocks.

The preparation in combinatorial chemistry library and screening are well known to those skilled in the art.This type of combinatorial chemistry library includes but not limited to, peptide library (see, such as, United States Patent (USP) 5,010,175, the people such as Furka, Int.J.Pept.Prot.Res.37:487-493 (1991) and Houghton, Nature 354:84-88 (1991)).Also other can be used for generation of the chemical method of chemical diversity libraries.This type of chemical method includes but not limited to, intends peptide (such as PCT International Publication WO 93/20242), random biological oligomer (such as PCT International Publication WO 92/00091), the benzodiazepine of peptide (peptoid) (such as PCT International Publication WO 91/19735), coding (such as U.S. Patent number 5,288,514), diversomers such as hydantoins, benzodiazepine with the dipeptides (people such as Hobbs, Proc.Nat.Acad.Sci.USA 90:6909-6913 (1993)), vinylogous polypeptide (vinylogous the polypeptide) (people such as Hagihara, J.Amer.Chem.Soc.114:6568 (1992)), there is the non-peptide analoglike peptide (people such as Hirschmann of glucose support, J.Amer.Chem.Soc.114:9217-9218 (1992)), analog organic synthesis (the people such as Chen of little library of compounds, J.Amer.Chem.Soc.116:2661 (1994)), few carbamate (the people such as Cho, Science 261:1303 (1993)), and/or the peptidyl phosphonate ester (people such as Campbell, J.Org.Chem.59:658 (1994)), nucleic acid library is (see Ausubel, Berger and Sambrook, all see above), peptide nucleic acid library (see, such as United States Patent (USP) 5, 539, 083), antibody library (see, the people such as such as Vaughn, Nature Biotechnology, 14 (3): 309-314 (1996) and PCT/US96/10287), carbohydrate libraries (see, the people such as such as Liang, Science, 274:1520-1522 (1996) and United States Patent (USP) 5, 593, 853), organic molecule library (see, such as benzodiazepine , Baum C & EN, January 18, the 33rd page (1993), isoprenoid, United States Patent (USP) 5,569,588, thiazolidone and a Buprofezin, United States Patent (USP) 5,549,974, pyrrolidine, United States Patent (USP) 5,525,735 and 5,519,134, morpholinium compound, United States Patent (USP) 5,506,337, benzodiazepine , 5,288,514 etc.).

Equipment for the preparation of combinatorial libraries can obtain from commercial channel (see, such as, 357MPS, 390MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050Plus, Millipore, Bedford, MA).In addition, a lot of combinatorial libraries itself can obtain from commercial channel (see, such as, ComGenex, Princeton, N.J., Tripos, Inc., St.Louis, MO, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD etc.).

F. the method for the correctives of nerve degeneration biomarker is identified

Generally be similar to wild type STAT3 and TYK2 sequence for the nerve degeneration biomarker polypeptide of Screening test and polynucleotide and keep at least one biologically active of albumen.Optionally, the fragment of full length sequence will be comprised, such as kinase domain or transactivation domain for the nerve degeneration biomarker polypeptide of determination of activity or polynucleotide.Polypeptide of the present invention or its domain will be covalently attached to heterologous protein to produce the chimeric protein be used in mensuration described herein.When polypeptide of the present invention has 110% relative to contrast, be optionally 150%, 200%, 300%, 400%, 500% or the activity value of 1000-2000% time, it is activated.

Use is recombinated or naturally occurring polypeptide measures the candidate modulator of nerve degeneration biomarker activity.Albumen can be separation, in cells, expression in the film being derived from cell, organizing or expressing in animal, can be restructuring or naturally occurring.Such as, can using-system section, dissociate cell (such as coming from the tissue of expressing polypeptide of the present invention), conversion cell or film.One of external or in vivoassay method described herein is used to test suppression situation.

The combination of test compounds and polypeptide of the present invention, domain or chimeric protein can in the solution, in duplicature, be attached to solid phase, measure in lipid monolayer or in vesica.Can use such as spectroscopic properties (such as fluorescence, absorption value, refractive index), fluid mechanics (such as shape), chromatogram or solubility characteristics change to detect described test compounds in conjunction with situation.

By the sample processed through potential correctives (such as, " test compounds ") or measure and do not contain the degree that the control sample of test compounds compares to detect adjustment.Control sample (without candidate compound process) is assigned to the relative activity value of 100.When the activity value relative to contrast is about 90%, optional 50%, during optional 25-0%, reach the suppression to polypeptide of the present invention.

Additionally provide the Screening test of the compound of the expression for regulating nerve degeneration biomarker polynucleotide and polypeptide.Screening technique generally comprises the mensuration of carrying out based on cell, wherein, test compounds and one or more is expressed the cells contacting of STAT3 or TYK2, then detects increase or the minimizing (transcript or translation product) of expression.The cell of any expression STAT3 or TYK2 polypeptide can be used, such as neuronal cell, carry out described mensuration.

Expression can be detected by multitude of different ways.As described above, the expression of nerve degeneration biomarker polynucleotide can measure like this: use with the probe of nerve degeneration biomarker transcript (or by its derivative complementary nucleic acid) specific hybrid to detect the mRNA of cells.Or, immunological method can be used to detect nerve degeneration biomarker polypeptide, such as such mensuration: use the antibody of specific binding polypeptide to detect cell lysate.

Reporter molecule system also can be used for the correctives identifying nerve degeneration biomarker expression.The cell of number of different types can be used in reporter assay.The cell of not endogenous expression nerve degeneration biomarker polypeptide can be protokaryon, but preferably eucaryon.Eukaryotic can be any routine for generation of the cell of cell with recombinant nucleic acid construct.Exemplary eukaryotic includes but not limited to, HCN-1 (cortical neuron), 1321N1 (astrocytoma), H4 (glioma), IMR32 and PC12 clone.

Can arrange various contrast to guarantee that the activity observed is believable, this comprises use and does not carry out parallel reactor containing the cell of reporter constructs, or is not contacted with test compounds by the cell carrying reporter constructs.

Multiple in vitro and in vivo determination method can be used to assess activity, thus measurement function, chemistry and physical influence.When TYK2, these determination methods comprise the catalytic phosphatase monitoring such as substrate.Provide exemplary kinase assays in an embodiment.Or, can by substrate and TYK2 polypeptide be being contained ³²incubation in the damping fluid of P-γ ATP the amount measuring the substrate of phosphorylation are to measure TYK2 by the ability of substrate (such as STAT3) phosphorylation.

Can also type of service be the mensuration of high flux purposes.Such as, kinase catalytic γ-phosphoryl is transferred to suitable hydroxyl acceptor from ATP, and discharges proton.Therefore, use the damping fluid/indicator system of suitably coupling based on the mensuration detecting this proton can be used for detection of active (see, such as Chapman & Wong Bioorg Med Chem 10:551-5,2002).

Or the cutting of the caspase 3 that TYK2 and STAT3-can be used to mediate and natural death of cerebral cells are to measure activity.In this type of measures, detect the mark of natural death of cerebral cells, such as DNA fragmentation, cells viability.The determination method being suitable for high flux screening form can be used to detect cells viability, and such as colourimetry or fluorescence viability measure.Such as, Alamar blue (AB) measures, and introduce redox indicator, it changes color or fluorescence in response to metabolic activity.When there is living cells, Alamar blue fluoresces; When there is dead cell, then do not fluoresce.Or can be read this type of by flow cytometry in microtiter plate to measure.Colorimetric estimation such as MTT determination method (its measure MTT (3-(4,5-dimethyl) thiazol-2-yl-2,5-diphenyltetrazoliumbromide bromine salt is reduced to formazan) also can be advantageously used in high throughput format, to measure cells viability and propagation.

Other mensuration measuring cell number can also be used.These comprise and measure dyestuff and mix determination method in cell DNA.The amount of the dyestuff mixed is direct and cell number is proportional.Such as, the dyestuff of such as Hoechst 33342 can be used cell dyeing, and it is incorporated in the DNA of living cells, determines cell number by the amount measuring fluorescence.Can also directly count cell.

Or, the intracellular signaling event in TYK2 and STAT3 downstream can be used to measure activity.Such as, technology described herein can be used to measure the gene expression (such as iNOS, IRF-1) activated by STAT3 transactivation.Or, the cutting of Caspase-3 can be detected, described in embodiment.

Those compounds identified at first by any aforementioned screening technique can be tested further to verify apparent activity.Preferably, suitable animal model is used to carry out this type of research.Whether the citation form of these class methods comprises, and is applied in the lead compound of qualification in initial screening to the animal being used as mankind AD model, then measure AD and be really conditioned and/or improve.For verifying that the animal model of research is generally the mammal of any kind.The instantiation of suitable animal includes but not limited to, primate, Mouse and rat.Exemplary animal model for AD is APP/PS1 mouse.

G. embodiment

1. embodiment 1: in the brain tissue of Alzheimer disease (AD) model mice, STAT3 raises in the phosphorylation of Tyr705

In order to identify the signal transduction path related in AD illness, screen expression and the functional status of multiple signal transduction molecule in AD mouse brain.The lysate of the different piece (comprising cortex, hippocampus and cerebellum) of brain tissue is prepared from APP/PS1 transgenic mice [APP (Swe)/PS1 (Δ E9)] (at 9 monthly ages, it is the clear AD mouse model characterized).Find that the tyrosine phosphorylation level of STAT3 consistance in the cortex of AD mouse brain raises (Figure 1A).These find to show to there is association between the pTyr-STAT3 level raised and AD illness.

The tyrosine phosphorylation (Figure 1B) of the STAT3 of rising is detected in the cortex of 4 months to 18 months large AD mouse.Reported these AD mouse and had amyloid-beta (A β) deposition when ~ 6 to 7 monthly age, it be the principal ingredient of AD brain person in middle and old age spot, before this shows that the tyrosine phosphorylation of STAT3 occurs in formation senile plaque expelling.

In the cortex of wild-type mice, almost can't detect the tyrosine phosphorylation of STAT3, and in the cortex of APP/PS1 mouse, observe the remarkable rising of the STAT3 of tyrosine phosphorylation.Pass through immunostaining, we find neuron (> 80%) location altogether of activated (tyrosine phosphorylation) STAT3 and NeuN positive, but do not locate altogether with the Deiter's cells of the GFAP-positive, this shows that the activation of STAT3 mainly occurs in the neuron of AD mouse brain further.In addition, find that the generation region of tyrosine phosphorylation in AD mouse brain of STAT3 is similar to the region (dyeed by A β and indicate) of senile plaque expelling generation.

2. the tyrosine phosphorylation of STAT3 in the beta induced cortical neuron of embodiment 2:A

The Cortical Neurons of Rat cultivated is used to study the effect of STAT3 tyrosine phosphorylation in AD as cell culture model.Use A β peptide 25-35 process cortical neuron (7DIV).Observe the remarkable rising of the tyrosine phosphorylation of STAT3, and continued phosphorylation (Fig. 2) in all 4 hours of A β process.The tyrosine phosphorylation of the STAT3 that immunostaining display is induced by A β process concentrates in the neuron of the 'beta '-tubulin III-positive.

3. embodiment 3:STAT3 strike low or that STAT3 is active suppression neuroprotective unit cell from the beta induced natural death of cerebral cells of A

In order to study the effect in the Neuronal cell death of STAT3 in AD, STAT3siRNA is used to strike the expression of STAT3 in low PC12 cell.Struck low about 80% STAT3 albumen (Fig. 3 A).After 4 days break up (being induced by NGF), use A β peptide process cell.Striking of STAT3 lowly decreases the beta induced cell death of A (Fig. 4 A).In addition, the suppression (by using specificity STAT3 inhibitor process cell) that STAT3 activates reduces the level (Fig. 3 B) of the Caspase-3 of the beta induced cutting of A, this indicates neuronal cell and adjusts the degree of dying.Caspase-3 is intracellular cysteine proteinases, and it participates in neuronal cell tune and dies.Its as the tune for cellular damage die response a part be activated.To sum up, these find to show that STAT3 activity has vital role in the Neuronal cell death of A β inducing peptide.

4. embodiment 4:Tyk2 mediates the tyrosine phosphorylation of the beta induced STAT3 of A, and be the beta induced neuronal cell of A adjust die required.

Due to the tyrosine phosphorylation of the beta induced STAT3 of A, so next target is the upstream regulation agent of qualification STAT3.Different inhibitor pre-service cortical neurons was used before A β process.JAK inhibitor 1 completely inhibit (its multiple tyrosine kinase for STAT3 upstream comprises JAK1, JAK2, JAK3 and Tyk2 and has strong inhibition effect) tyrosine phosphorylation (Fig. 4 A) of the beta induced STAT3 of A.But the inhibitor AG490 of JAK2 does not then show the depression effect being similar to JAK inhibitor 1.These find to illustrate that the phosphorylation of the STAT3 that A is beta induced is mediated by the JAK family member except JAK2.In addition, Src inhibitors of kinases PP2 and protein synthesis inhibitor hexanoyl imines do not affect the phosphorylation of the beta induced STAT3 of A, and this shows the phosphorylation of the STAT3 that A is beta induced: (i) is not undertaken by Src approach; (ii) albumen is not needed to synthesize.Use A β peptide process cortical neuron 1 hour.By specific antibody by endogenous Tyk2 or JAK2 immunoprecipitation, detect sediment by the antibody 4G10 of monoclonal phosphorylation-tyrosine.Different from JAK2, Tyk2 tyrosine phosphorylation (Fig. 4 B) can be detected in the cell of A β process.

Then have detected Tyk2 and adjust the participation situation in dying at the neuronal cell that A is beta induced.From Tyk2 ^-/-mouse prepares Primary cortical neurons culture.In contrast neuron, the process of A β peptide causes STAT3 at the tyrosine phosphorylation of Tyr705.But, at Tyk2 ^-/-in neuron, this pecific phosphorylation reduces (Fig. 4 C).In addition, at Tyk2 ^-/-in neuron, Caspase-3 activation beta induced by A and neuronal cell are adjusted to die and are also reduced.To sum up, Tyk2 is required for the beta induced STAT3 phosphorylation of A, and the beta induced Caspase-3 of its mediation A cuts and Neuronal cell death.

5. embodiment 5: the A β process induction iNOS gene expression in cortical neuron

Whether affecting the transcriptional activity of STAT3 in order to characterize A β peptide further, using and being connected to Luciferase construct (pSTAT3-Luc) the transfection PC12 cell that STAT3-responds enhancer element.Be connected to STAT1-and respond the Luciferase construct (pGAS-Luc) of enhancer element with comparing.Consistent with former report, NGF process is induction of the promoter activity of STAT3 and STAT1, but A β peptide only induces STAT3 promoter activity (Fig. 5 A).

With A β peptide process Primary cortical neurons 6 hours.The mRNA level in-site of nitric oxide synthase type (iNOS) is detected by PCR in real time.INOS is one of nitricoxide synthase (NOS) of 3 types in mammal, and it is responsible for producing gas signal transduction molecule nitrogen monoxide (NO).INOS has vital role in the immune response of inflammation and pathogen-inducible.After being exposed to some cell factor such as interferon-γ (IFN-γ), iNOS produces NO, and it carries out intracellular signaling by JAK family and stat protein.The STAT activated is Dimerized and be indexed into nucleus, and they increase the expression of transcription factor such as IRF-1 there, and IRF-1 is bonded to the specific DNA element of iNOS gene promoter region then to increase the expression of iNOS gene.

After the process of A β peptide, iNOS gene expression dose raises.What is interesting is, this rising of iNOS gene expression can be blocked by the pre-service of STAT3 inhibitor or JAK inhibitor I, and this shows that JAK-STAT approach is required (Fig. 5 B) for the rise of the beta induced iNOS gene expression of A.

6. embodiment 6:STAT3 tyrosine phosphorylation is visible in AD brain section

In order to determine the tyrosine phosphorylation whether observing STAT3 in the brain of AD patient, the hippocampus brain section from 4 AD patients is dyeed.All 4 patients demonstrate similar dyeing pattern.The tyrosine phosphorylation of STAT3 is clearly observed in AD brain section.Pass through immunostaining, we also find neuron (about 70%) location altogether of active STAT3 and the NeuN positive, but do not locate altogether with the Deiter's cells (about 7%) of the GFAP-positive, this and the result from APP/PS1 transgenic mice brain section are consistent (Fig. 6).

7. method

Preparation and the western blot of brain extract, cell extract: from the transgenic mice of different phase (from 2 months to 18 months), be separated 4 brains divide, comprise cortex, hippocampus, cerebellum and corpus straitum, and remain on dry ice.By freezing brain tissue homogenate in the homogenate buffer (25mM Tris-HCl, pH 7.4,150mM NaCl, 1mM EDTA, pH 7.4,50mM NaF) with multiple protein enzyme inhibitor.Retain supernatant and be used for western blot, use RIPA lysis buffer (150mM NaCl, 1% (v/v) Nonidet P-40,0.5% deoxycholic aicd, 0.1% (w/v) SDS; (wherein again extract sediment containing multiple protein enzyme inhibitor (2 μ g/ml Aprotinins, 1mM Phenylmethylsulfonyl fluorine, 5mM benzamidine, 1mM sodium orthovanadate (NaOV) and 10 μ g/ml soybean trypsin inhibitors).Collect cortical neuron or the PC12 cell of original cuiture, and cracking in the RIPA lysis buffer containing protease inhibitors.

Immunohistochemical analysis: paraffin brain section is boiled 5 minutes, to carry out antigen recovery in 1mM edta buffer liquid (pH 8.0).DAB or AEC dyeing is carried out according to the instructions of manufacturer.For fluorescence immunization coloration, use in room temperature, containing 4% lowlenthal serum in the PBS of 0.4%Triton X-100, brain section is closed 20 minutes.Primary antibodie (anti-NeuN is used at 4 DEG C; 1: 200) and anti-pSTAT3 (Tyr705) (1: 100) section is incubated overnight, then anti-rabbit IgG (Invitrogen, 1: the 1000) incubation that the anti-mouse using Alexa Fluor 488-to put together in room temperature and Alexa Fluor 568-put together 1 hour.Then washing slice use anti-color fading agent (anti-fade reagent) (Invitrogen) to carry out mounting, then analyzes under Olympus Laser Scanning Confocal Microscope (Fluoview BX61, Olympus).Use 4% paraformaldehyde, in room temperature, the cortical neuron of cultivation is fixed 30 minutes, then use and close 20 minutes containing 4% lowlenthal serum in the PBS of 0.4%Triton X-100 in room temperature.Use anti-NeuN monoclonal antibody (1: 500) and P-Tyr STAT3 polyclonal antibody (1: 200) to carry out immunohistochemical analysis at 4 DEG C to spend the night.Then the anti-mouse using Alexa Fluor 488 or Alexa Fluor 568-to put together in room temperature or anti-rabbit IgG (Invitrogen, 1: 1000) were by cell marking 1 hour.Then use anti-color fading agent (Invitrogen) that neuron is carried out mounting, and analyze under Olympus Laser Scanning Confocal Microscope (Fluoview BX61, Olympus).

Cells viability: according to instructions (the cell proliferation reagent box I of manufacturer, Roche), by cell by tetrazolium salts (3-(4,5-dimethylthiazole-2-base)-2,5-diphenyltetrazoliumbromide bromine salt (MTT) ability that is reduced to coloured formazan product carrys out quantify cellular viability.In brief, in cell, add (1/10 of culture volume) MTT reagent (5mg/ml, in DPBS).At 37 DEG C by cell culture 4 hours.The MTT solubilization buffer (0.01M HCl, 10%SDS) of 2 times of volumes is added, then at 37 DEG C of incubations 24 hours in the dark in cell.Plate reader (EYNEX REVELATION 4.22, MRC TC REVELATIONDYNEX TECHNOLOGIES) is used to measure absorption value at 570nm.Living cells is only had to take in MTT.Cells viability is expressed as the percent of absorption value relative to the absorption value of control cultures of the cell of A β process.

Caspase-3 determination of activity: use CaspACE ^tMassay System, Fluorometric kit (Promega) measures the activity of Caspase-3.In brief, by lysis, and measured the activity of Caspase-3 in cell extract by fluorometry.(excitation wavelength is 360nm to use SpectraMaxGemini; Emission wavelength is 460nm) fluorescent emission of AMC that produces of the specificity cutting that measures the peptide substrate DEVD marked due to Caspase-3 couples of fluorophore AMC.Protein determination is carried out according to correction contrast.

Quantitative RT-PCR: according to the instructions of manufacturer, uses RNeasy trace quantity reagent kit (Qiagen) to be separated total serum IgE from the cortical neuron of A β process.Random six conjuncted (random hexamer) (Roche) are used to synthesize Article 1 cDNA chain.Use Bio-RadMX3000P real-time PCR system, use the Auele Specific Primer of target specific gene to carry out quantitative PCR.

Should be appreciated that embodiment described herein and the embodiment object just to explaining, its various modification or to change be known for those skilled in the art, and in the spirit and scope being included in the application and the scope of claim of enclosing.The all publication quoted herein, patent and patented claim are incorporated to herein by way of reference in full in order to all objects.

Claims (35)

1., for the identification of the method for the compound of regulation and control Neurodegenerative conditions, said method comprising the steps of:

I compound contacts with STAT3 or TYK2 polypeptide by (); With

(ii) functional effect of this compound for STAT3 or TYK2 is measured, wherein compound comprises the suppression of the phosphorylation at 705 tyrosine places of STAT3 for the functional effect of STAT3, and wherein said functional effect indicates this compound modulates Neurodegenerative conditions.

2. the process of claim 1 wherein that the feature of described Neurodegenerative conditions is the deposition of 4 amyloid in brain.

3. the method for claim 1, wherein said Neurodegenerative conditions is selected from Alzheimer disease (AD), Parkinson's, repeats of dentatorubropallidolatrophy atrophy disease (DRPLA), transparent inclusion disease (NIHID) in neuronal kernel, dementia with Lewy body, Tang's grace syndrome, Hallervorden Spatz disease, prion disease, Argyrophilic grain dementia, cortical basal degeneration, dementia pugilistica, dispersivity neurofibrillary tangles, GSSShi is sick, Hallervorden Spatz disease, Creutzfeldt-Jakob disease, Niemann-Pick disease 3 type, stein-leventhal syndrome, subacute sclerosing panencephalitis, spinocerebellar ataxia, Huntington disease, pik disease and repeats of dentatorubropallidolatrophy atrophy disease.

4. the process of claim 1 wherein that described polypeptide is restructuring.

5. the process of claim 1 wherein and measure described functional effect in vitro.

6. the process of claim 1 wherein and measure described functional effect in eukaryotic.

7. the method for claim 6, wherein said cell is neuronal cell.

8. the method for claim 6, the tune wherein by measuring cell is died and is measured described functional effect.

9. the method for claim 6, wherein measures described functional effect by measuring transcribing of STAT3 activation.

10. the phosphorylation at 705 the tyrosine places that the process of claim 1 wherein by mensuration STAT3 measures described functional effect.

11. the process of claim 1 wherein that described functional effect is the phosphorylation suppressing STAT3.

12. the process of claim 1 wherein and measure described functional effect by measuring TYK2 kinase activity.

13. the process of claim 1 wherein that described compound is for the specific antibody of STAT3 or TYK2 or antibody fragment.

14. the process of claim 1 wherein that described compound is for the specific inhibitor peptides of STAT3 or TYK2.

15. the process of claim 1 wherein that described compound is organic molecule.

16., for the identification of the method for compound of regulation and control Alzheimer disease, said method comprising the steps of:

I the polynucleotide of compound with coding STAT3 or TYK2 polypeptide contact by (); With

(ii) functional effect of this compound for STAT3 or TYK2 is measured, the phosphorylation of 705 tyrosine or the expression of TYK2 polypeptide wherein by measuring STAT3 measure described functional effect, and wherein said functional effect indicates this compound modulates Alzheimer disease.

The method of 17. claims 16, wherein the feature of Alzheimer disease is the deposition of 4 amyloid in brain.

The method of 18. claims 16, wherein said polynucleotide are restructuring.

The method of 19. claims 16, wherein measures described functional effect in vitro.

The method of 20. claims 16, wherein measures described functional effect in eukaryotic.

The method of 21. claims 20, wherein said cell is neuronal cell.

The method of 22. claims 20, the tune wherein by measuring cell is died and is measured described functional effect.

The method of 23. claims 20, wherein measures described functional effect by measuring transcribing of STAT3 activation.

The method of 24. claims 16, the expression wherein by measuring STAT3 or TYK2 measures described functional effect.

The method of 25. claims 24, wherein uses immunoassay to measure the expression of STAT3 or TYK2.

The method of 26. claims 24, wherein measures the expression of STAT3 or TYK2 by RT-PCR.

The method of 27. claims 16, wherein said compound is for the specific antisense molecule of STAT3 or TYK2.

The method of 28. claims 16, wherein said compound is for the specific RNAi molecule of STAT3 or TYK2.

The purposes of 29. reagent being determined at the level of the STAT3 polypeptide of 705 tyrosine place phosphorylations in the diagnosticum for the preparation of the Neurodegenerative conditions in diagnosis individuality, described diagnosis comprises the following steps:

I () measures the level available from the STAT3 polypeptide 705 tyrosine place phosphorylations in the biological sample of described individuality;

(ii) compare in the level of the STAT3 705 tyrosine place phosphorylations in the sample of step (i) and normal biological specimen in the level of the STAT3 of 705 tyrosine place phosphorylations, thus diagnose the Neurodegenerative conditions in described individuality.

The purposes of 30. claims 29, wherein determination step comprises immunoassays.

The purposes of 31. claims 29, the feature of wherein said Neurodegenerative conditions is the deposition of 4 amyloid in brain.

The purposes of 32. claims 29, wherein said Neurodegenerative conditions is selected from Alzheimer disease (AD), mild cognitive impairment, Parkinson's, repeats of dentatorubropallidolatrophy atrophy disease (DRPLA), transparent inclusion disease (NIHID) in neuronal kernel, dementia with Lewy body, Tang's grace syndrome, Hallervorden Spatz disease, prion disease, Argyrophilic grain dementia, cortical basal degeneration, dementia pugilistica, dispersivity neurofibrillary tangles, GSSShi is sick, Hallervorden Spatz disease, Creutzfeldt-Jakob disease, Niemann-Pick disease 3 type, stein-leventhal syndrome, subacute sclerosing panencephalitis, spinocerebellar ataxia, Huntington disease, pik disease and repeats of dentatorubropallidolatrophy atrophy disease.

The method of 33. claims 7 or 21, wherein said neuronal cell is adult's cortical neuron or hippocampal neuron.

The method of 34. claims 16, the functional effect of wherein said compound comprises the expression of the polynucleotide suppressing coding STAT3 or TYK2 polypeptide.

35. the purposes of claim 29, wherein show that this individuality is in the risk of Neurodegenerative conditions in the increase of the level of the STAT3 polypeptide of 705 tyrosine place phosphorylations.